Biomimetic drug delivery of an immunomodulatory agent for the treatment of ocular conditions

ABSTRACT

A method for treating an ocular disorder in a subject comprising administering a therapeutic agent-loaded carrier to an ocular site of the subject in need thereof, wherein the therapeutic agent loaded-carrier provides controlled delivery of the therapeutic agent under conditions suitable for recruiting regulatory T cells to an ocular region of interest or inducing regulatory T cells in an ocular region of interest.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.15/628,233, filed on Jun. 20, 2017, which claims priority to U.S.Application No. 62/353,908 filed on Jun. 23, 2016, which is incorporatedherein by reference in its entirety.

BACKGROUND

To the best of our knowledge, current ophthalmic drug delivery systemsdo not provide a long term release of microparticles containing animmunomodulatory agent for the treatment of inflammatory eye diseases.The treatments for dry eye disease are based upon the condition. Mildtreatments can include lifestyle changes, such as wearing sunglasses andless exposure to drying winds. Additional therapies to aid mild tomoderate inflammation are tear substitutes. Artificial tears do providetemporary relief for patients; however, most formulations containpreservatives such as benzalkonium chloride that can cause eyeirritation and hyperosmolarity of the tear film. Also, anti-inflammatorytreatments are used for patients with severe inflammation. Thesetreatments have shown to decrease inflammation in patients, however areonly intended for short-term use, and long-term application has beenimplicated in conditions such as glaucoma and retinopathy. Othertreatment approaches include, tear duct plugs, which reduce tearturnover. However, plugs do not address the underlying cause of theinflammatory disease.

Dry eye disease (DED) is a multifactorial ocular condition,characterized by inflammation of the ocular surface and tear filminstability, which afflicts as many as 1 in 5 individuals globally.Individuals with DED suffer symptoms including blurred vision,foreign-body and/or burning sensation, light sensitivity, and in severecases, corneal ulcerations leading to vision loss. Current treatmentspredominantly address the symptoms of DED and include artificial tears,punctual occlusion with tear plugs, and ophthalmic corticosteroids.Artificial tear substitutes may provide temporary relief for patients;however, most artificial tear formulations contain preservatives, suchas benzalkonium chloride, which sometimes can cause tear filmhyperosmolarity. This adverse effect can trigger death ofmucin-producing goblet cells, leading to further ocular irritation. Tearplugs may be used with or without artificial tears to reduce tearturnover by occluding the draining tear duct; however, these punctalplugs must be inserted by a physician, and limitations include issueswith plug retention and increased risk of ocular infections. Even withregular use of artificial tears and/or punctual plugs, many patientsremain symptomatic because these palliative treatments do not addressthe underlying cause of DED. Recently, a number of studies demonstratedan inflammatory basis for DED, which led to the application of topicalcorticosteroids to treat DED. While ophthalmic corticosteroids broadlysuppress ocular inflammation and can alleviate symptoms of DED, theeffects are transient and are prescribed for short-term use.Consequently, treatment of DED typically requires long-term use ofcorticosteroids, which is associated with severe side effects, such assteroid-induced glaucoma and retinopathy. Furthermore, despitesuppressing production of inflammatory mediators, corticosteroids do notaddress the underlying imbalance between pro-inflammatory immune cellsand immunosuppressive cells.

In DED, infiltration of pathogenic pro-inflammatory CD4⁺ T cells causesa breakdown in immunological homeostasis, ultimately compromising thelacrimal functional unit (LFU), which includes the cornea, conjunctiva,lacrimal glands, meibomian glands, and the interconnecting innervation.As T cells proliferate in the ocular tissues, these cells secretepro-inflammatory cytokines, such as IFN-γ, which inhibit naturallysuppressive immune cells known as regulatory T cells (Tregs). Thisultimately leads to a shift in the immunological balance betweentissue-protective Tregs and tissue-destructive pro-inflammatory(effector) T cells. Since the importance of Tregs contributing toimmunological tolerance has become evident over the years, investigatorshave examined methods to utilize these immunosuppressive cells. Notably,adoptive transfer of Tregs from mice with DED can suppress inflammationin a T-cell deficient nude mouse administered effector T-cells from aDED mouse. Moreover, due to the low population of Tregs found in thehuman body (5-15%), application of ex vivo transfer of Tregs has beenproposed as a method of therapeutic modulation in order to enhance thelimited numbers of Tregs. Despite such evidence suggesting thatenhancing Treg populations ex vivo is a viable therapeutic approach,there are many hurdles associated with translating a cellular-therapy tothe clinic. These include expansion, contamination, and the potentialhazard of Tregs differentiating into conventional T cells.

SUMMARY

Disclosed herein are methods for treating an ocular disorder in asubject comprising administering a therapeutic agent-loaded carrier toan ocular site of the subject in need thereof, wherein the therapeuticagent loaded-carrier provides controlled delivery of the therapeuticagent under conditions suitable for recruiting regulatory T cells to anocular region of interest or inducing regulatory T cells in an ocularregion of interest.

Further disclosed herein are methods for treating an ocular disorder ina subject comprising administering a therapeutic agent-loaded carrier toan ocular site of the subject in need thereof, wherein the therapeuticagent loaded-carrier provides controlled delivery of the therapeuticagent, the therapeutic agent is selected from CCL22, interleukin 2,rapamycin, transforming growth factor beta (TGF-β), retinoic acid, orvasoactive intestinal peptide (VIP), and the ocular disorder is dry eyedisease, uveitis, allergic conjunctivitis, scleritis, or Age-RelatedMacular Degeneration (AMD).

Additionally disclosed herein are methods for treating an oculardisorder in a subject comprising administering a therapeuticagent-loaded carrier to an ocular site of the subject in need thereof,wherein the therapeutic agent loaded-carrier provides controlleddelivery of the therapeutic agent under conditions suitable foractivating regulatory T cells in an ocular region of interest.

Also disclosed herein is a composition comprising therapeuticagent-loaded microparticles, wherein the therapeutic agent is selectedfrom CCL22, interleukin 2, rapamycin, transforming growth factor beta(TGF-β), retinoic acid, or vasoactive intestinal peptide (VIP), and thecomposition does not include a hydrogel.

The foregoing will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic timeline for CCL22 releasing PLGA microparticlesfor preventing Dry eye Disease (DED) in mice. Anti-GITR was injected viai.p. 5 days prior to administration of Concanavalin A and CCL22 MPs intothe lacrimal gland.

FIGS. 2A-2C: CCL22 microparticles prevent DED symptoms in mice. (FIG.2A) The treatment group of CCL2 MP does not significantly decrease tearproduction as compared to the experimental groups of ConA/Blank MP,which was significantly higher than ConA and equivalent to salinetreatment (n=8) shown as mean±S.D. (FIG. 2B) The treatment group of CCL2MP was able to maintain integrity of the epithelial layer of the corneasignificantly than the ConA/Blank MP and ConA groups, and similar to thetreatment group of saline alone, as observed by corneal fluoresceinstaining (FIG. 2C) A clinical score of the ocular surface staining,demonstrates that ConA/CCL22 MP treatment significantly decreases thepermeability of the cornea as compared to ConA/Soluble CCL22, andConA/Blank MP experimental groups (n=8) as shown as mean±S.D. *p≤0.05;** p≤0.01; *** p≤0.001

FIG. 3: CCL22 MPs prevent reduction of goblet cells in the conjunctiva.Representative histology images (20×) of PAS (Periodic Acid Schiff)murine conjunctiva. Significance was calculated using a One-Way Anovafollowed by Bonferroni post-hoc test * p≤0.05;** p≤0.01; *** p≤0.001

FIGS. 4A-4D: CCL22 Microparticles suppress T effector type of cells inthe regional draining lymph nodes. (FIG. 4A) Flow cytometry performed onthe lymph nodes suggests the Saline MP treatment group has a significantpercentage of infiltrating CD4+ lymphocytes in the cervical lymph nodesas compared to the diseased (ConA) (n=8) shown as mean±S.D. (FIG. 4B)CD4+ IFN-γ+ cells were analyzed to experimental groups (n=8) as shown asmean±S.D. (FIG. 4C) FoxP3+ Tregs was analyzed by flow cytometry in thelymph nodes (n=8) as shown as mean±S.D. (FIG. 4D) Foxp3− cells aresignificantly reduced in the cervical lymph nodes of the CCL22 MP groupas compared to the ConA and ConA/Blank MP (n=8) as shown as mean±S.D.Significance was calculated using a One-Way Anova followed by Bonferronipost-hoc test * p≤0.05;** p≤0.01; *** p≤0.001

FIGS. 5A-5D: CCL22 MPs enhance anti-inflammatory responses in thelacrimal gland. (FIG. 5A) How cytometry performed on the lymph nodessuggests the CCL22 MP treatment group has a significant reduction ofinfiltrating CD4+ IFN-γ+ lymphocytes in the intraorbital lacrimal glandas compared to the diseased (ConA) and ConA/Blank MP (n=8) shown asmean±S.D. (FIG. 5B) Percentage of Tregs as determined by flow cytometry(n=8) as shown as mean±S.D. (FIG. 5C) Intracellular cytokine stainingwas determined after use of a cell stimulation cocktail to detect IL-10expressing Tregs (FIG. 5D) The ratio of Tregs/T effectors was determinedby CD4+ Foxp3+ to CD4+ IFN-γ+ cells. (n=8) shown as mean±S.D.Significance was calculated using a One-Way Anova followed by Bonferronipost-hoc test * p≤0.05; ** p≤0.01; *** p≤0.001

FIGS. 6A-6D: Characterization of engineered Porous microparticles loadedwith the chemokine CCL22 and blank microparticles. FIG. 6A) SEM ofPorous blank microparticles (1000×). FIG. 6B) SEM of porousmicroparticles with CCL22 encapsulated (1000×). FIG. 6C) CoulterCounter: Average Volume Impedance measurements of Microparticles. FIG.6D) Release Kinetics of porous CCL22 Microparticles

FIG. 7: Flow Cytometry of Inguinal Lymph Nodes. Total CD4+ cells in theinguinal lymph nodes show a significant difference in the Anti-GITRCCL22 MP group as compared to ConA/CCL22 MP

FIG. 8 is a representation of the application of CCL22 microparticles torecruit functional regulatory T cells to the eye.

FIG. 9. TRI microspheres for the prevention of inflammation associatedwith Dry eye Disease (DED) in mice. A timeline for the experimentalmurine model of inflammation induce via Concanavalin A.

FIGS. 10A-10C. TRI MS prevent clinical signs of inflammation associatedwith DED (FIG. 10A) Wetting of phenol red threads were measured inmillimeters using a dissecting microscope (n=6) shown as mean±S.D. (FIG.10B) Representative images of histological sections of the eyes (20×)were quantified to identify differences in the Treg-inducing MS groupcompared to the diseased groups and non-diseased group (100 μm scalebar). (FIG. 10C) Goblet cells shown are the pink/purple (Periodic AcidSchiff stained) cells located in the conjunctiva labeled with arrows andthe groups are shown as mean±S.D. * p≤0.05;** p≤0.01; *** p≤0.001, ****p≤0.0001.

FIGS. 11A-11B. TRI MS reduce ocular surface staining (FIG. 11A)Representative images of corneal fluorescein staining. (FIG. 11B)Clinical corneal fluorescein staining scores of the ocular surface on ascale of (0-4) (n=6) shown as mean±S.D. (n=6). * p≤0.05;** p≤0.01

FIG. 12. Administration of TRI MS reduces levels of cytokines in thelacrimal gland shown as mean±SEM. * p≤0.05

FIG. 13A-13D. Representative lacrimal gland fixed frozen cryosectionsstained for T-cells (CD3⁺ T cells—Cyan), Regulatory T-cells (FoxP3⁺ Tcells—Red), and nuclei (DAPI-blue). Scale bars are 100 μm

FIGS. 14A-14I. Characterization of Treg-inducing Microspheres (FIG. 14A)Representative Scanning electron microscopy (SEM) image of Rapamycinmicrospheres (1000×) (FIG. 14B) Representative SEM image of IL-2Microspheres (1000×) (FIG. 14C) Representative image of TGF-β1Microspheres. (FIG. 14D) Release Kinetics of Rapamycin Microspheres isshown (n=3) (FIG. 14E) Release Kinetics of porous IL-2 Microspheres(n=3) (FIG. 14F) Release Kinetics of TGF-β1 Microspheres (n=3). (FIG.14G) Size distribution of Rapamycin Microspheres (FIG. 14H) Sizedistribution of IL-2 Microspheres (FIG. 14I) Size Distribution of TGF-β1Microspheres

DETAILED DESCRIPTION Terminology

The following explanations of terms and methods are provided to betterdescribe the present compounds, compositions and methods, and to guidethose of ordinary skill in the art in the practice of the presentdisclosure. It is also to be understood that the terminology used in thedisclosure is for the purpose of describing particular embodiments andexamples only and is not intended to be limiting.

An “animal” refers to living multi-cellular vertebrate organisms, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. Similarly, the term “subject”includes both human and non-human subjects, including birds andnon-human mammals, such as non-human primates, companion animals (suchas dogs and cats), livestock (such as pigs, sheep, cows), as well asnon-domesticated animals, such as the big cats.

The term “co-administration” or “co-administering” refers toadministration of an agent disclosed herein with at least one othertherapeutic or diagnostic agent within the same general time period, anddoes not require administration at the same exact moment in time(although co-administration is inclusive of administering at the sameexact moment in time). Thus, co-administration may be on the same day oron different days, or in the same week or in different weeks. In certainembodiments, a plurality of therapeutic and/or diagnostic agents may beco-administered by encapsulating the agents within the microparticlesdisclosed herein.

“Inhibiting” refers to inhibiting the full development of a disease orcondition. “Inhibiting” also refers to any quantitative or qualitativereduction in biological activity or binding, relative to a control.

“Microparticle”, as used herein, unless otherwise specified, generallyrefers to a particle of a relatively small size, but not necessarily inthe micron size range; the term is used in reference to particles ofsizes that can be, for example, administered to the eye in the form ofan eye drop that can be delivered from a squeeze nozzle container, andthus can be less than 50 nm to 100 microns or greater. In certainembodiments, microparticles specifically refers to particles having adiameter from about 1 to about 25 microns, preferably from about 10 toabout 25 microns, more preferably from about 10 to about 20 microns. Inone embodiment, the particles have a diameter from about 1 to about 10microns, preferably from about 1 to about 5 microns, more preferablyfrom about 2 to about 5 microns. As used herein, the microparticleencompasses microspheres, microcapsules, microparticles, microrods,nanorods, nanoparticles, or nanospheres unless specified otherwise. Amicroparticle may be of composite construction and is not necessarily apure substance; it may be spherical or any other shape.

“Ocular region” or “ocular site” means any area of the eye, includingthe anterior and posterior segment of the eye, and which generallyincludes, but is not limited to, any functional (e.g., for vision) orstructural tissues found in the eyeball, or tissues or cellular layersthat partly or completely line the interior or exterior of the eyeball.Ocular regions include the anterior chamber, the posterior chamber, thevitreous cavity, the choroid, the suprachoroidal space, the subretinalspace, the conjunctiva, the subconjunctival space, the episcleral space,the intracorneal space, the epicorneal space, the sclera, the parsplana, surgically-induced avascular regions, the macula, the retina, andthe lacrimal functional unit (LFU), which includes the cornea,conjunctiva, lacrimal glands, meibomian glands, and the interconnectinginnervation.

“Ocular condition” means a disease, ailment or condition which affectsor involves the eye or one of the parts or regions of the eye. Broadlyspeaking the eye includes the eyeball and the tissues and fluids whichconstitute the eyeball, the periocular muscles (such as the oblique andrectus muscles) and the portion of the optic nerve which is within oradjacent to the eyeball.

A “therapeutically effective amount” refers to a quantity of a specifiedagent sufficient to achieve a desired effect in a subject being treatedwith that agent. Ideally, a therapeutically effective amount of an agentis an amount sufficient to inhibit or treat the disease or conditionwithout causing a substantial cytotoxic effect in the subject. Thetherapeutically effective amount of an agent will be dependent on thesubject being treated, the severity of the affliction, and the manner ofadministration of the therapeutic composition. For example, a“therapeutically effective amount” may be a level or amount of agentneeded to treat an ocular condition, or reduce or prevent ocular injuryor damage without causing significant negative or adverse side effectsto the eye or a region of the eye

“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop, or administering a compound or composition to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing a pathology or condition,or diminishing the severity of a pathology or condition. As used herein,the term “ameliorating,” with reference to a disease or pathologicalcondition, refers to any observable beneficial effect of the treatment.The beneficial effect can be evidenced, for example, by a delayed onsetof clinical symptoms of the disease in a susceptible subject, areduction in severity of some or all clinical symptoms of the disease, aslower progression of the disease, an improvement in the overall healthor well-being of the subject, or by other parameters well known in theart that are specific to the particular disease. The phrase “treating adisease” refers to inhibiting the full development of a disease, forexample, in a subject who is at risk for a disease such as dry eyedisease.

“Preventing” a disease or condition refers to prophylactic administeringa composition to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping a pathology or condition, or diminishing the severity of apathology or condition. In certain embodiments, “treating” meansreduction or resolution or prevention of an ocular condition, ocularinjury or damage, or to promote healing of injured or damaged oculartissue

“Pharmaceutical compositions” are compositions that include an amount(for example, a unit dosage) of one or more of the disclosed compoundstogether with one or more non-toxic pharmaceutically acceptableadditives, including carriers, diluents, and/or adjuvants, andoptionally other biologically active ingredients. Such pharmaceuticalcompositions can be prepared by standard pharmaceutical formulationtechniques such as those disclosed in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. (19th Edition).

The terms “pharmaceutically acceptable salt or ester” refers to salts oresters prepared by conventional means that include salts, e.g., ofinorganic and organic acids, including but not limited to hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonicacid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid,tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid,maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelicacid and the like. “Pharmaceutically acceptable salts” of the presentlydisclosed compounds also include those formed from cations such assodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and frombases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)aminomethane, andtetramethylammonium hydroxide. These salts may be prepared by standardprocedures, for example by reacting the free acid with a suitableorganic or inorganic base. Any chemical compound recited in thisspecification may alternatively be administered as a pharmaceuticallyacceptable salt thereof. “Pharmaceutically acceptable salts” are alsoinclusive of the free acid, base, and zwitterionic forms. Descriptionsof suitable pharmaceutically acceptable salts can be found in Handbookof Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH(2002). When compounds disclosed herein include an acidic function suchas a carboxy group, then suitable pharmaceutically acceptable cationpairs for the carboxy group are well known to those skilled in the artand include alkaline, alkaline earth, ammonium, quaternary ammoniumcations and the like. Such salts are known to those of skill in the art.For additional examples of “pharmacologically acceptable salts,” seeBerge et al., J. Pharm. Sci. 66:1 (1977).

“Pharmaceutically acceptable esters” includes those derived fromcompounds described herein that are modified to include a carboxylgroup. An in vivo hydrolysable ester is an ester, which is hydrolysed inthe human or animal body to produce the parent acid or alcohol.Representative esters thus include carboxylic acid esters in which thenon-carbonyl moiety of the carboxylic acid portion of the ester groupingis selected from straight or branched chain alkyl (for example, methyl,n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example,methoxymethyl), aralkyl (for example benzyl), aryloxyalkyl (for example,phenoxymethyl), aryl (for example, phenyl, optionally substituted by,for example, halogen, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy) or amino);sulphonate esters, such as alkyl- or aralkylsulphonyl (for example,methanesulphonyl); or amino acid esters (for example, L-valyl orL-isoleucyl). A “pharmaceutically acceptable ester” also includesinorganic esters such as mono-, di-, or tri-phosphate esters. In suchesters, unless otherwise specified, any alkyl moiety presentadvantageously contains from 1 to 18 carbon atoms, particularly from 1to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Anycycloalkyl moiety present in such esters advantageously contains from 3to 6 carbon atoms. Any aryl moiety present in such esters advantageouslycomprises a phenyl group, optionally substituted as shown in thedefinition of carbocyclyl above. Pharmaceutically acceptable esters thusinclude C₁-C₂₂ fatty acid esters, such as acetyl, t-butyl or long chainstraight or branched unsaturated or omega-6 monounsaturated fatty acidssuch as palmoyl, stearoyl and the like. Alternative aryl or heteroarylesters include benzoyl, pyridylmethyloyl and the like any of which maybe substituted, as defined in carbocyclyl above. Additionalpharmaceutically acceptable esters include aliphatic L-amino acid esterssuch as leucyl, isoleucyl and especially valyl.

For therapeutic use, salts of the compounds are those wherein thecounter-ion is pharmaceutically acceptable. However, salts of acids andbases which are non-pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound.

The pharmaceutically acceptable acid and base addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid and base addition salt forms which the compounds are ableto form. The pharmaceutically acceptable acid addition salts canconveniently be obtained by treating the base form with such appropriateacid. Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds containing an acidic proton may also be converted intotheir non-toxic metal or amine addition salt forms by treatment withappropriate organic and inorganic bases. Appropriate base salt formscomprise, for example, the ammonium salts, the alkali and earth alkalinemetal salts, e.g. the lithium, sodium, potassium, magnesium, calciumsalts and the like, salts with organic bases, e.g. the benzathine,N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids suchas, for example, arginine, lysine and the like.

The term “addition salt” as used hereinabove also comprises the solvateswhich the compounds described herein are able to form. Such solvates arefor example hydrates, alcoholates and the like.

The term “quaternary amine” as used hereinbefore defines the quaternaryammonium salts which the compounds are able to form by reaction betweena basic nitrogen of a compound and an appropriate quaternizing agent,such as, for example, an optionally substituted alkylhalide, arylhalideor arylalkylhalide, e.g. methyliodide or benzyliodide. Other reactantswith good leaving groups may also be used, such as alkyltrifluoromethanesulfonates, alkyl methanesulfonates, and alkylp-toluenesulfonates. A quaternary amine has a positively chargednitrogen. Pharmaceutically acceptable counterions include chloro, bromo,iodo, trifluoroacetate and acetate. The counterion of choice can beintroduced using ion exchange resins.

Disclosed herein are methods and compositions for treating oculardisorders. Illustrative disorders include, but are not limited to, dryeye disease, uveitis, allergic conjunctivitis, scleritis and Age-RelatedMacular Degeneration (AMD). In certain embodiments, the disorder is aninflammatory mediated ocular disorder, particularly in cases withchronic inflammation as an underlying cause.

The methods include administering a therapeutic agent-loaded carrier toa subject. The carrier may be in the form of a thin film, a rod, contactlens, or microparticles. In certain embodiments, the compositionsinclude therapeutic agent-loaded microparticles.

Illustrative therapeutic agents include the natural, protein chemokine,C-C chemokine motif (CCL22), interleukin 2 (IL-2), rapamycin,transforming growth factor beta (TGF-β), retinoic acid, and vasoactiveintestinal peptide (VIP).

In certain embodiments, the therapeutic agents are highly effective atrecruiting Tregs, which is essential to the maintenance of immunologicalhomeostasis to ensure the prevention of chronic inflammation andautoimmunity. In particular, CCL22, retinoic acid and VIP are effectiveat recruiting Tregs.

In certain embodiments, the therapeutic agents are highly effective atinducing Tregs, which is essential to the maintenance of immunologicalhomeostasis to ensure the prevention of chronic inflammation andautoimmunity. In particular, IL-2, rapamycin and TGF-β are effective atinducing Tregs.

In certain embodiments, the therapeutic agent-loaded microparticles arehighly effective at recruiting a specific type of T cell called aregulatory T-cell (Tregs), which is essential to the maintenance ofimmunological homeostasis to ensure the prevention of chronicinflammation and autoimmunity. The dissolvable microspheres areeffective at recruiting endogenous Treg cells to a local site, naturallyresolving inflammatory symptoms.

In certain embodiments, the method disclosed herein establish atherapeutic agent gradient to recruit Tregs to restore tissue damage inocular disorders such as DED. For example, disclosed herein areTreg-recruiting CCL22 microparticles (CCL22 MPs) that when injected inthe lacrimal gland increase the ratio of Tregs to CD4⁺ IFN-γ⁺ cells inthe gland and reduce proliferation of CD4⁺ lymphocytes in the regionaldraining lymph nodes suggesting that the reduction of effector T cellsleads to the prevention of DED. Furthermore, CCL22 MPs prevent clinicalsigns of DED through the maintenance of aqueous tear production,preservation of goblet cell density, and corneal fluorescein staining.Thus, controlled release of CCL22 is able to recruit endogenous Tregs torestore tear film stability and ocular surface health.

Current topical therapeutics focus on providing agents acting asantagonists to hinder a specific cell involved in the pathogenesis ofinflammatory eye diseases. However, long-term use of anti-inflammatoryagents can cause retinopathy and glaucoma. The use of the microparticlesdisclosed herein is an approach to restore the homeostatic balanceinstead of solely targeting a pathogenic cell. Therefore, this treatmentdisclosed herein will resolve the underlying etiology of the disease,particularly dry eye disease. The methods and compositions disclosedherein also could result in a dramatic increase of patient complianceand reduce disease morbidity.

In one embodiment, disclosed herein are therapeutically-relevant,modular platforms to deliver therapeutic agents in vivo by artificialparticles into the vicinity of Tregs. In one embodiment, the deliveredagents modulate Treg cell proliferation. In one embodiment, thedelivered factors modulate Treg cell immunosuppressive capacity.

In one embodiment, the method comprises introducing artificialmicroparticles in vivo wherein Tregs are recruited and/or activated. Inone embodiment, the Treg cell recruitment and/or activation inducesbiological homeostasis thus resolving the ocular disease or condition.

In certain embodiments, the ocular disorder is treated via the inductionof a subject's own Tregs from naïve CD4⁺ T cells. This approach utilizesthe body's own natural mechanism to differentiate peripheral naïve CD4⁺T cells into Tregs through a subset of antigen presenting cells known astolerogenic dendritic cells (tDCs). Specifically, tDCs can inducedifferentiation of Tregs through the secretion of IL-2 and TGF-βcytokines. However, the maintenance of Tregs is somewhat more complexand depends on a local microenvironment that is not only favorable todifferentiation of Tregs, but also unfavorable to differentiation intoother effector T cells. One-way to ensure that cells do notdifferentiate into pathogenic effector T cells is through the smallmolecule, rapamycin. Rapamycin (Rapa) is an mTOR inhibitor that cansuppress the generation and proliferation of effector T cells.

In certain embodiments, the body's own endogenous Tregs are enriched bydelivering a combination of Treg inducing factors through TRImicrospheres (TGF-β1, Rapamycin (Rapa), and IL-2). This localdrug-delivery system is able to increase the prevalence of Tregs and, inturn, prevent key signs of dry eye disease such as aqueous tearsecretion, conjunctival goblet cells, epithelial corneal integrity, andreduce the pro-inflammatory cytokine milieu in the tissue.

In certain embodiments, the amount of agent loaded into themicroparticles may range from 1 ng to 1 mg, more particularly 1 to 100μg, and most particularly, 20 to 30 μg agent per mg of microparticles.In certain specific embodiments, the amount of agent loaded into themicroparticles is 25-30 μg agent per mg of microparticles.

The polymers for the microparticle may be bioerodible polymers so longas they are biocompatible. Preferred bio-erodible polymers arepolyhydroxyacids such as polylactic acid and copolymers thereof.Illustrative polymers include poly glycolide, poly lactic acid (PLA),and poly (lactic-co-glycolic acid) (PLGA). Another class of approvedbiodegradable polymers is the polyhydroxyalkanoates.

Other suitable polymers include, but are not limited to: polyamides,polycarbonates, polyalkylenes, polyalkylene glycols, polyalkyleneoxides, polyalkylene terephthalates, polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,polyglycolides, polysiloxanes, polyurethanes and copolymers thereof,alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, celluloseesters, nitro celluloses, polymers of acrylic and methacrylic esters,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxylethyl cellulose, cellulose triacetate,cellulose sulphate sodium salt, poly(methyl methacrylate),poly(ethylmethacrylate), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexylmethacrylate),poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,polypropylene polyethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), poly(vinyl acetate), poly vinylchloride polystyrene, polyvinylpryrrolidone, alginate,poly(caprolactone), dextran and chitosan.

The percent loading of an agent may be increased by “matching” thehydrophilicity or hydrophobicity of the polymer to the agent to beencapsulated. In some cases, such as PLGA, this can be achieved byselecting the monomer ratios so that the copolymer is more hydrophilicfor hydrophilic drugs or less hydrophilic for hydrophobic drugs.Alternatively, the polymer can be made more hydrophilic, for example, byintroducing carboxyl groups onto the polymer. A combination of ahydrophilic drug and a hydrophobic drug can be encapsulated inmicroparticles prepared from a blend of a more hydrophilic PLGA and ahydrophobic polymer, such as PLA.

A preferred polymer is a PLGA copolymer or a blend of PLGA and PLA. Themolecular weight of PLGA is from about 10 kD to about 80 kD, morepreferably from about 10 kD to about 35 kD. The molecular weight rangeof PLA is from about 20 to about 30 kDa. The ratio of lactide toglycolide is from about 75:25 to about 50:50. In one embodiment, theratio is 50:50.

Illustrative polymers include, but are not limited to,poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid to glycolicacid ratio, M_(n)=10 kDa, acid-terminated, referred to as 502H);poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid to glycolicacid ratio, M_(n)=25 kDa, acid-terminated, referred to as 503H);poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid to glycolicacid ratio, M_(n)=30 kDa, acid-terminated, referred to as 504H);poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid to glycolicacid ratio, M_(n)=35 kDa, ester-terminated, referred to as 504); andpoly(D,L-lactic-co-glycolic acid) (PLGA, 75:25 lactic acid to glycolicacid ratio, M_(n)=10 kDa, referred to as 752).

In certain embodiments, the polymer is an ester-terminated PLGA.

In certain embodiments, the polymer is a polyethyleneglycol-poly(lactic-co-glycolic acid) copolymer.

In certain embodiments, the polymer microparticles are biodegradable.

In certain embodiments, the agent-loaded microparticles may have avolume average diameter of 200 nm to 30 μm, more particularly 1 to 10μm. In certain embodiments, the agent-loaded microparticles do not havea volume average diameter of 10 μm or greater since such largerparticles are difficult to eject from a container in the form of an eyedrop. The agent-loaded microparticles may be pore less or they maycontain varying amounts of pores of varying sizes, typically controlledby adding NaCl during the synthesis process.

In certain embodiments, the agent-loaded microparticle-containingcomposition does not include a hydrogel, particularly a thermoresponsivehydrogel.

The agent-loaded microparticle fabrication method can be single ordouble emulsion depending on the desired encapsulated agent solubilityin water, molecular weight of polymer chains used to make themicroparticles (MW can range from ˜1000 Da to over 100,000 Da) whichcontrols the degradation rate of the microparticles and subsequent drugrelease kinetics.

The microparticle disclosed herein may provide for sustained release ofan agent. For example, the sustained release may be over a period of atleast one day, more particularly at least 5 days or at least 10 days,and most particularly at least 30 days. The agent release can be linearor non-linear (single or multiple burst release). In certainembodiments, the agent may be released without a burst effect. Forexample, the sustained release may exhibit a substantially linear rateof release of the therapeutic agent in vivo over a period of at leastone day, more particularly at least 5 days or at least 10 days, and mostparticularly at least 30 days. By substantially linear rate of releaseit is meant that the therapeutic agent is released at a rate that doesnot vary by more than about 20% over the desired period of time, moreusually by not more than about 10%. It may be desirable to provide arelatively constant rate of release of the agent from the deliverysystem over the life of the system. For example, it may be desirable forthe agent to be released in amounts from 0.1 to 100 μg per day, moreparticularly 1 to 10 μg per day, for the life of the system. However,the release rate may change to either increase or decrease depending onthe formulation of the polymer microparticle. In certain embodiments,the delivery system may release an amount of the therapeutic agent thatis effective in providing a concentration of the therapeutic agent inthe eye in a range from 1 ng/ml to 200 μg/ml, more particularly 1 to 5μg/ml. In certain embodiments, there is no initial lag phase of release.The desired release rate and target drug concentration can varydepending on the particular therapeutic agent chosen for the drugdelivery system, the ocular condition being treated, and the subject'shealth.

The microparticle disclosed herein may provide for controlled release ofan agent. The term “controlled release” as used herein, refers to theescape of any attached or encapsulated factor at a predetermined rate.For example, a controlled release of an agent may occur resulting fromthe predicable biodegradation of a polymer particle (i.e., for example,an artificial antigen presenting cell). The rate of biodegradation maybe predetermined by altering the polymer composition and/or ratioscomprising the particle. Consequently, the controlled release may beshort term or the controlled release may be long term. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year.

In certain embodiments the agent-loaded microparticles may be includedin a composition suitable for topical administration in the form of aliquid eye drop. The eye drop(s) may be administered to any ocularstructure. The eye drops may be self-administered by the subject. Theeye drop will conform comfortably to the conjunctival sac and releasethe loaded agent. The eye drop may be administered on a regimen whereinthe interval between successive eye drops is greater than at least oneday (although in certain embodiments the eye drop may be administeredonce daily or more than once daily). For example, there may be aninterval of at least 5 days, at least one week, or at least one monthbetween administrations of an eye drop(s). The agent-loadedmicroparticles disclosed herein drastically decreases the dosingfrequency (thereby increasing the likelihood of patient compliance andrecovery/prevention of worsening symptoms), it does so while avoidingclinician involvement for administration by being completelynoninvasive.

The microparticle-containing composition disclosed herein may include anexcipient component, such as effective amounts of buffering agents, andantioxidants to protect a drug (the therapeutic agent) from the effectsof ionizing radiation during sterilization. Suitable water solublebuffering agents include, without limitation, alkali and alkaline earthcarbonates, phosphates, bicarbonates, citrates, borates, acetates,succinates and the like, such as sodium phosphate, citrate, borate,acetate, bicarbonate, carbonate and the like. These agents areadvantageously present in amounts sufficient to maintain a pH of thesystem of between about 2 to about 9 and more preferably about 4 toabout 8. As such the buffering agent may be as much as about 5% byweight of the total system. Suitable water soluble preservatives includesodium bisulfite, sodium bisulfate, sodium thiosulfate, ascorbate,benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuricacetate, phenylmercuric borate, phenylmercuric nitrate, parabens,methylparaben, polyvinyl alcohol, benzyl alcohol, phenylethanol and thelike and mixtures thereof. These agents may be present in amounts offrom 0.001 to about 5% by weight and preferably 0.01 to about 2% byweight.

In certain embodiments, the microparticles disclosed herein may beadministered via injection. Injection sites include but are not limitedto intraorbital lacrimal gland, extraorbital lacrimal gland,intraorbital injection, subconjunctival, intravitreal, posterior andanterior chambers of the eye.

Recent experimental and clinical investigations into theimmunopathogenesis of DED suggest that the disorder is primarilymediated by CD4⁺ T cells, which cause aberrant inflammation thatcontributes to ocular surface damage and tear film instability. Morespecifically, inflammation of the lacrimal gland leads to insufficientsecretion of aqueous tear production resulting in clinical signs of DED.In order to mediate the development and perpetuation of inflammation,there is a sophisticated repertoire of CD4⁺ T cells that maintainimmunological tolerance and tissue homeostasis to ensure the preventionof chronic inflammation and autoimmunity. Specifically, this criticalsubset of T lymphocytes known as regulatory T cells (Tregs) are involvedin stabilizing the immune microenvironment of the eye and activelyregulating inflammation caused by an immune response mitigated viaeffector T cells.

The approach disclosed herein recruits endogenous Treg populations atthe site of inflammation in order to promote an immunological balancebetween T effectors and Tregs ultimately preventing clinical signs ofDED. In order to test the efficacy of the CCL22 MPs, an experimentalmurine dry eye disease model that induces inflammation usingConcanavalin A (ConA), which nonspecifically expands and activates Tcells through cross-linking the T cell receptor (FIG. 1), can beemployed. Particularly, this murine model was selected, because DED hasbeen thought to be primarily mediated by T cells.

Typically, inflammation associated with DED is characterized by symptomsof reduced tear film stability. We observed that administration of CCL22MPs was able to effectively prevent the reduction in aqueous tearproduction seen in mice with ConA-induced DED (FIG. 2A). Notably, theprotective/restorative effects on tear production required that CCL22 besustainably released from MPs, as soluble CCL22 was unable to preventloss of tear production (FIG. 2A). Studies suggest this could be due tothe requirement of a chemokine concentration gradient to directlymphocyte migration. While sustained release of CCL22 from degradableMPs can establish such a gradient for several days (e.g, at least twodays) to weeks (e.g., at least two weeks), a bolus of CCL22 wouldquickly diffuse, forming a uniform concentration distribution. With thereduction of tear film ConA-induced DED mice, the lack of ocular surfacelubrication leads to loss of corneal epithelial cells and the breakdownof intracellular tight junctions leading to an increase of cornealepithelial permeability. Ultimately, this allows fluorescein dye todiffuse into tissue spaces left by resident desquamated epithelialcells. Clinical implications corresponding to a decrease of cornealintegrity can affect visual acuity. Interestingly, the CCL22 MPs wereable to maintain ocular surface health, whereas ConA-induced DED mice(with or without Blank MPs) possessed statistically higher cornealfluorescein staining scores (FIGS. 2B and 2C), which traditionally is aclinical readout for corneal epitheliopathy. Together, the data suggestthat CCL22 MPs are able to prevent clinical signs of DED inducedinflammation directly correlating to disease severity.

In DED, an increase of inflammation corresponds directly to conjunctivalgoblet cell apoptosis. To confirm that CCL22 MPs were maintaining gobletcell health, we analyzed histological sections of the conjunctiva inorder to identify goblet cell density. Goblet cells secrete mucin, acomponent found in the tear film, which protect the ocular surface. Asinflammation occurs to the ocular surface, the composition of the tearfilm is altered leading to a reduction of goblet cells and an increaseof corneal fluorescein staining. We observed a decrease in the overalldensity of the mucin-filled goblet cells in mice with ConA-induced DED,relative to non-diseased (Saline) mice, which is consistent with cornealfluorescein images (FIG. 3). Loss of goblet cells might be due to higherlevels of the Th1 cytokine (IFN-γ). Increased amounts of IFN-γ/IL-13cytokines inhibit the promotion of goblet cell differentiation. Morespecifically, IFN-γ antagonizes production of IL-13, thus promotingapoptosis and squamous metaplasia of the goblet cells in theconjunctival epithelium. Notably, administration of CCL22 MPs maintainedgoblet cell density, as seen in the representative histological imagescompared to the diseased, diseased blank microparticles and solubleCCL22 (FIG. 3). Given that a loss of goblet cell density has shown toresult from production of IFN-γ, we investigated lymphocyte populationsin the regional draining lymph nodes and lacrimal gland, lookingspecifically at IFN-γ⁺ T effectors and FoxP3⁺ Tregs. We examined thelymph nodes, because this region serves as a reservoir for immune cellsthat can migrate to the ocular surface. Specifically, cervical lymphnodes, which drain from the eye, are critical sites for the induction ofocular surface inflammation through the activation of T effectors (FIG.4). Moreover, these effector T cells migrate via chemokines such as CCR6and CXCR3 from the cervical lymph nodes (CLN) to the ocular surface.Thus, we analyzed phenotypic expression of CD4⁺ T lymphocytes in theregional draining superficial CLN. As expected with a T-cell mitogen,administration of ConA possessed significantly higher total CD4⁺ T cellpopulations in the CLN. Furthermore, we identified increases of CD4⁺IFN-γ⁺Th1-type cells and activated T-effectors (CD4⁺CD25⁺FoxP3−) in theCLN of ConA-induced DED mice as compared to those treated with CCL22MPs. This effect might be mediated due to the migration of dendriticcells to the regional lymph nodes via the chemokine CCR7, which activatepathogenic T cells that cause ocular surface and corneal inflammation.As immune cells migrate from the lymph nodes to the lacrimal gland,pro-inflammatory cytokines are secreted by the immune cells thatperpetuate inflammation and negatively affect the function of the gland.Remarkably, as we examined the gland, data suggest the CCL22 MPsincreased the amount of CD4⁺ FoxP3⁺ IL-10⁺ Tregs in the lacrimal glandand ultimately shifted the ratio towards suppression resulting in agreater Treg/T effector balance compared to the diseased, and ConA+BlankMPs.

To demonstrate that CCL22 MP suppression of DED is mediated via Tregs,we used a GITR agonist (anti-DTA-1) to inhibit/block the suppressiveeffects of Tregs on other T cells. Anti-GITR modulates regulatory Tcells through directly abrogating their suppressive function whileco-stimulating other conventional T cells. The ability of anti-GITR tosuppress Treg function and worsen clinical evaluations of DED isconsistent with recent work that demonstrated the GITR ligand plays anintegral role in ocular immunity regulating inflammation via theparticipation of photoreceptors. Additionally, administration ofanti-GITR negated the therapeutic effects of CCL22 MP treatment, withreduced tear production (FIG. 2A) and increased corneal fluoresceinstaining (FIG. 2B, C). A significant influx of CD4⁺ T cells occurred inthe regional draining lymph nodes on anti-GITR compared to the all thegroups (FIG. 4). In fact, this corresponded to an increase in thefrequency of CD4⁺ IFN-γ⁺ Th1 cells and a decrease in the frequency ofIL-10 producing Tregs in the lacrimal gland, compromising theimmunological balance between T effectors and Tregs as compared to theadministration of CCL22 MPs (FIG. 4C). Overall, anti-GITRreverses/neutralizes the effects of CCL22MPs, and in some cases yieldsgreater pro-inflammatory cell populations than ConA alone.

Thus, we demonstrated that CCL22 MPs are capable of preventinginflammation in DED suggesting the potential broad applicability of thischemokine delivery system. Here we focused on an experimental murinemodel to prevent clinical signs of DED.

Several Illustrative Embodiments are Described Below in FollowingNumbered Clauses

1. A method for treating an ocular disorder in a subject comprisingadministering a therapeutic agent-loaded carrier to an ocular site ofthe subject in need thereof, wherein the therapeutic agentloaded-carrier provides controlled delivery of the therapeutic agentunder conditions suitable for recruiting regulatory T cells to an ocularregion of interest.

2. The method of clause 1, wherein the regulatory T cells areendogenous.

3. The method of clause 1, wherein the ocular disorder is dry eyedisease, uveitis, allergic conjunctivitis, scleritis, or Age-RelatedMacular Degeneration (AMD).

4. The method of clause 1, wherein the therapeutic agent is selectedfrom CCL22, interleukin 2, rapamycin, transforming growth factor beta(TGF-β), retinoic acid, or vasoactive intestinal peptide (VIP).

5. The method of clause 1, wherein the ocular disorder is dry eyedisease and the therapeutic agent is CCL22.

6. The method of clause 1, wherein the therapeutic agent-loaded carrieris in the form of therapeutic agent-loaded microparticles.

7. The method of clause 1, wherein the therapeutic agent-loaded carriercomprises therapeutic agent-loaded microparticles.

8. The method of clause 6, wherein the microparticles comprise poly(lactic-co-glycolic acid).

9. The method of clause 1, wherein the method provides controlledrelease under conditions to provide a concentration gradient within ornear the ocular disorder site.

10. The method of clause 6, wherein the therapeutic agent-loadedmicroparticles are included in a composition that does not include ahydrogel.

11. The method of clause 5, wherein the therapeutic agent-loaded carrieris in the form of therapeutic agent-loaded microparticles.

12. The method of clause 11, wherein the microparticles comprise poly(lactic-co-glycolic acid).

13. A composition comprising therapeutic agent-loaded microparticles,wherein the therapeutic agent is selected from CCL22, interleukin 2,rapamycin, transforming growth factor beta (TGF-β), retinoic acid, orvasoactive intestinal peptide (VIP), and the composition does notinclude a hydrogel.

14. The composition of clause 13, wherein the microparticles comprisepoly (lactic-co-glycolic acid).

EXAMPLES Example 1—CCL22

Fabrication of Microparticles—Poly (lactic-co-glycolic) acid (PLGA)microparticles encapsulating recombinant mouse CCL22 (R&D Systems,Minneapolis Minn.) were made according to a previously reportedwater-oil-water double emulsion technique. Glowacki, A. J. et al. Proc.Natl. Acad. Sci. U.S.A 110, 18525-30 (2013). Briefly, 200 mg of RG502Hpoly(D,L-lactide-co-glycolide) polymer (Sigma Aldrich, St. Louis, Mo.)was dissolved in 4 ml of dichloromethane and vortexed. Then 2004, of anaqueous solution containing 5 μg of recombinant mouse CCL22 and 2 mg ofBSA (Sigma Aldrich, St. Louis, Mo.) with 15 mmol NaCl was pipetted intothe mixture of polymer and dichloromethane. Next, the first water-in-oilemulsion was prepared by sonicating the polymer and DCM solution with150 μL of deionized water and 50 μL of CCL22 solution for a period of 10seconds. Then the second water-oil emulsion was created by homogenizing(L4RT-A, Silverson) first water-oil emulsion with 60 mL 2% (wt./vol)polyvinyl alcohol (molecular weight ˜25,000 g/mol, 98 mole % hydrolyzed;PolySciences) for a period of 60 seconds at 3,000 rpms. The homogenizedsolution was then mixed with 1% polyvinyl alcohol and placed onto a stirplate for 3 hours in order for the dichloromethane to evaporate. Themicroparticles were then collected and washed with deionized water inorder to remove any remaining residual polyvinyl alcohol. Lastly, themicroparticles were placed in 5 mL of deionized water, frozen in liquidnitrogen, and lyophilized for 72 hours (VirTis BenchTop K freeze dryer).The microparticles were stored at −20° C.

Characterization of Microparticles—Morphology of CCL22MPs wascharacterized using scanning electron microscopy (JEOL, JSM-6330F,Peabody, Mass.) and volume impedance measurements were completed on aBeckman Coulter Counter (Multisizer-3, Beckman Coulter, Fullerton,Calif.). In vitro release assays were completed by incubating 10 mg ofCCL22 MPs in 1 ml of phosphate buffered saline (PBS), placed on arotator at 37° C. Release media (supernatant) was sampled periodicallyand CCL22 concentrations were quantified using an enzyme-linkedimmunosorbent assay (ELISA) (R&D Systems, Minneapolis, Minn.).

Mice—Six to eight-week-old female Balb/c mice. (Charles RiversLaboratories, Wilmington, Mass.). The Institutional Animal Care and UseCommittee approved the protocol, and all of the animals used in theexperiments were treated according to the Association for Research inVision and Ophthalmology Statement for the Use of Animals in Ophthalmicand Vision Research.

Experimental DED Model and Treatments—To induce DED, mice wereanesthetized, and 200 μg of ConA (Sigma Aldrich, St. Louis, Mo.) in 20μl of saline was injected into each intraorbital lacrimal gland (2 permouse) with a 28.5 gauge needle using a dissecting microscope. DEDtreatments included Blank (empty) or CCL22 MPs (25 mg/ml), which weremixed with ConA or saline and injected. (Olympus SZX10, Waltham, Mass.).Soluble CCL22 was injected with ConA at 2.5 μg in 20 μl. To inhibit Tregfunction in vivo, anti-GITR (DTA-1) (BioXCell, Lebanon, N.H.) wasinjected i.p. (500 μg per mouse) five days prior to the injection ofConA and CCL22 MPs.

Measurement of Tear Production—Aqueous tear production was measured withphenol red cotton threads (Oasis Medical, San Dimas, Calif.). Thread wasplaced in the lateral canthus of the eye for a period of 60 seconds, andwetting was measured in millimeters using a dissecting microscope(Olympus SZX10, Waltham, Mass.).

Corneal Permeability—To evaluate the corneal epithelial layer,fluorescein stain (1 uL of 1% solution) was applied to the conjunctivalsac and 5 μl of saline was used to wash off any excess dye. The surfaceof the cornea was evaluated using a dissecting microscope with afluorescent excitation lamp (Olympus SZX10, Waltham, Mass.). Eyes wereevaluated in a masked fashion by an independent ophthalmologist, andscored 0 for no staining, score 1 for a quarter of staining, score of 2for less than a half, score of 3 for half, and 4 for more than half ofthe eye.

Histopathology—At the end of the study, eyeballs were exenterated thenharvested and fixed in formalin for 24 hours. Eyes were sectioned 5 μmthick and stained with Periodic Acid Schiff (PAS) to identify gobletcells in the conjunctiva. The histology section images were scannedusing a Zeiss Axio Scan. Z1 (Thornwood, N.Y.).

Immunophenotype Analysis by Flow Cytometry—Lacrimal glands and drainingcervical lymph nodes were harvested from the experimental murine groupsat the end of the study, and single cell suspensions were prepared.Cells were stained with the following fluorescent conjugated antibodies:anti-CD4 eFluor450 (RM4-5), anti-CD25 APC-Cy7 (PC61), anti-FoxP3 PE(FJK-16s) (eBioscience, San Diego, Calif. and BD Bioscience, San Jose,Calif.). For intracellular cytokine staining, the cells were placed in a96-well plate overnight in cell culture media with Cell StimulationCocktail (plus protein transport inhibitor) for a period of 12 hours andstained with anti-IL-10 APC (JES5-16E3), and anti-IFN-γ FITC (XMG 1.2)(eBioscience, San Diego, Calif.). Stained cells were analyzed with BDFACSDiva software, v6.1.3.

Statistical Analysis—Data expressed as mean±S.D. Comparisons betweenmultiple treatment groups were performed using one-way ANOVA, followedby Bonferroni multiple comparisons, and p<0.05 was consideredstatistically significant. Statistical tests were performed usingGraphPad Prism Software 6.0 (GraphPad Prism, San Diego, Calif.)

Results CCL22 MPs Prevent Clinical Symptoms of DED.

To assess whether CCL22 MPs could prevent clinical evaluationsassociated with DED, several aspects such as tear production, cornealfluorescein staining, and goblet cell density were examined One weekafter inducing DED, we hypothesized that ConA, the DED-inducing agent,would decrease tear production, while ConA plus treatment with CCL22 MPswould prevent a decrease of tear production. As expected, ConAsignificantly reduced tear secretion, relative to control mice (Salineinjection). Meanwhile, the effect of incorporating the CCL22 MPs witheither ConA or Saline was examined to determine if the chemokinemicroparticles were able to prevent a decrease in tear volume. CCL22 MPsand Blank (unloaded) MPs were injected (0.5 mg MPs/20 μl) with ConA todetermine if CCL22 MPs were able to prevent the clinical symptomsassociated with DED. The ConA+CCL22 MP group prevented tear productionloss to levels comparable to non-diseased (saline) mice, while treatmentwith ConA and ConA+Blank MP significantly reduced tear productionsimilarly (FIG. 2A) Additionally, we wanted to confirm that injectingmicroparticles does not contribute to symptoms associated with DED.Therefore, we examined the effects of blank microparticles in saline toidentify if they had an effect on tear secretion. As shown in FIG. 2A,the Saline Blank MPs possessed no significant difference in tearsecretion relative to the saline group at the conclusion of the study.CCL22 MP administration also appears to maintain the integrity of thecorneal epithelium. Corneal permeability, which is decreased with afaster rate of fluorescein elimination in healthy patients as comparedto those with DED, was visually evaluated by comparing uptake offluorescein staining on the corneal epithelium. Consistent with anintact/undamaged cornea, no staining was observed on the corneas ofcontrol mice (Saline and Saline Blank MP) after instillation offluorescein. In contrast, central punctate fluorescein staining wasobserved on corneas of the ConA, ConA+Blank MP, and ConA+Soluble CCL22groups (FIG. 2B). The fluorescein dye also appeared to stain the tearfilm in these groups. However, mice treated with ConA+CCL22 MPs showedno significant increase in corneal staining relative to non-diseased(Saline control) mice (FIG. 2C).

After one week, eyes were exenterated, and the conjunctiva was PeriodicAcid Schiff (PAS) stained to identify goblet cells interspersedthroughout the stratified squamous cells of the conjunctiva. Gobletcells play an important role in lubrication of the eye through theproduction of mucin, which is a component of the tear film. As a loss ofgoblet cell density occurs, tear film composition changes correlating toseverity of conjunctival disease. Treatment with ConA or ConA+ Blank MPled to a decrease in goblet cell density compared to the non-diseased(Saline or Saline+ Blank MP) control groups (FIG. 3). Notably, treatmentof DED (ConA) mice with CCL22 MPs restored goblet cell density to levelsseen in non-diseased mice (FIG. 3).

CCL22 MPs Suppress Proliferation of CD4⁺ T Cells in Regional DrainingLymph Nodes.

Previous investigations into DED have demonstrated ocular inflammationis mediated primarily by CD4+ T cells. Moreover, studies have identifiedan increased proliferation of T cells in the draining lymph nodes ofmice with DED. Additionally, it has been shown that CCL22 MPs canattract Tregs and skew T-cell populations locally. Glowacki, A. J. etal. Proc. Natl. Acad. Sci. U.S.A 110, 18525-30 (2013). Thus, todetermine whether CCL22 MP-mediated suppression of DED symptoms was dueto effects on the activation of T cells in the regional lymphoid tissue,we performed a phenotypic analysis of T-cell populations in the cervicallymph nodes (CLN). Total cells counts were included due to thesignificant increase in overall cell counts with the administration ofConA. CLN from mice with DED induced by ConA (with or without Blank MPs)exhibited significantly greater total numbers of CD4+ T cells (FIG. 3A).To further confirm the T-cell mediated immune response, we examined theexpression of the intracellular cytokine, IFN-γ. Results suggest thatCCL22 microparticles possess a decrease of CD4⁺IFN-γ⁺ cells (FIG. 3B).However, the diseased mice had significantly higher levels ofCD4⁺IFN-γ⁺(Th1) T cells (FIG. 3B), CD4⁺CD25⁺FoxP3⁺Tregs (FIG. 3C), andCD4⁺CD25⁺Foxp3− (Activated T-effectors) (FIG. 3D), than CLN from CCL22MPs and the non-diseased (Saline or Saline+Blank MP) control mice.Notably, CCL22 MP administration significantly reduced total numbers ofactivated T-effectors as compared to the diseased mice (FIG. 3D).Interestingly, there was a decrease in the total amount of Tregs.However, the decrease in Tregs was not as dramatic as the decrease ofIFN-γ⁺ cells for the CCL22 MP as compared to the diseased, ConA+BlankMP, and ConA+Soluble CCL22 (FIG. 3C). Finally, to confirm that therewere no systemic effects of ConA and/or CCL22MPs, we harvested distalinguinal lymph nodes, and performed a similar phenotypic analysis ofCD4⁺ T cells and CD4⁺CD25⁺FoxP3⁺ T cells (FIG. 7). We did observe asignificant difference in CD4⁺ lymphocyte count in the inguinal lymphnodes among any experimental groups, suggesting there is a systemicaffect due to the Anti-GITR. Results suggest that the CCL22 MPs have asignificantly lower population of IFN-γ producing T cells in the CLNrelative to the diseased control groups.

CCL22 MPs Reduce Infiltration of CD4⁺IFN-γ⁺ T cells in the LacrimalGland.

In DED, inflammation of the lacrimal gland increases the production ofpro-inflammatory cytokines, which results in lymphocyte homing andproliferation. Ultimately, the infiltration of lymphocytes and secretionof pro-inflammatory cytokines inhibit the normal function of the gland.To determine whether CCL22 MPs alter T-cell populations in the lacrimalgland, we harvested the intraorbital lacrimal glands and used flowcytometry to analyze T-cell populations in this tissue. Specifically, welooked at frequencies of pro-inflammatory CD4⁺ IFN-γ⁺ (Th1) T cells andCD4⁺ FoxP3⁺ Tregs in the gland. Lacrimal glands from mice with DEDinduced by ConA (w/or w/o Blank MP) had significantly greaterproportions of IFN-γ⁺ T cells (FIG. 5A) and significantly lowerproportions of FoxP3+Tregs (FIG. 5B), relative to non-diseased (salinew/or w/o Blank MP) mice. Notably, DED mice treated with ConA+CCL22 MPsshowed a significant reduction in the frequency of IFN-γ⁺ cells with aconcomitant increase in the frequency of FoxP3⁺Tregs (FIG. 5A,B),relative to ConA alone. A large proportion of the FoxP3⁺Tregs alsoexpressed the suppressive cytokine IL-10 (FIG. 5C). Ultimately, theshifts in pro-inflammatory cytokine, IFN-γ⁺ and anti-inflammatory Tregpopulations with CCL22 MPs contributed to a two-fold increase in theTreg/Th1-type ratio, relative to ConA alone (FIG. 5D) while theTreg/Th1-type ratio for the ConA+CCL22 MP group was still significantlyless than that for non-diseased (Saline or Saline+Blank MP) mice. Inaddition, we identified that there was a significant increase in thetotal amount of CD4⁺ T cells in the diseased mice and fewer total cellsin the CCL22 MP group. Specifically, CD4⁺IFN-γ⁺total cells were lower inthe CCL22 MP administered group. Collectively, the data suggest there isa significant reduction of T lymphocytes in the regional draining lymphnodes compared to the negative controls.

Administration of Anti-GITR Disables Regulatory T Cell Function.

To explore deeper the role of CCL22 MP recruitment of endogenous Tregsto reduce inflammation in the DED model, we wanted to confirm that thesuppressive effects suggested by our data were due to Tregs. Aglucocorticoid-induced tumor necrosis factor receptor (GITR) agonisticantibody (Anti-GITR) was administered 5 days before injection of ConAand CCL22 MPs (FIG. 1A). The outcome of anti-GITR essentially inhibitsthe ability of Tregs to suppress other T cells (non-Tregs). Importantly,anti-GITR injected before treatment with ConA and CCL22 MPs appeared toabrogate the symptom-reducing effects of CCL22 MPs, with aqueous tearproduction and punctate staining of the cornea comparable to (or worsethan) that of mice treated with ConA alone (FIG. 2A,B). Additionally,mice treated with anti-GITR before ConA+CCL22 MPs had significantlygreater numbers of total CD4⁺ T cells and activated T effectors, andcomparable numbers of IFN-γ⁺ Th1-type cells in the CLN, relative to micetreated with ConA alone (FIG. 4). Moreover, the lacrimal gland tissue ofmice treated with anti-GITR, ConA, and CCL22MP had similar frequenciesof CD4⁺IFN-γ⁺ T cells, FoxP3⁺ Tregs, and IL-10⁺ producing Tregs as micewith ConA-induced DED (FIG. 5). Taken together, these results indicatethat using anti-GITR to inhibit Treg-mediated suppression reverses thetherapeutic effects of CCL22 MPs in the DED model at the symptom andunderlying immunological levels.

Example 2—TRI Microspheres

Fabrication of Microspheres—TGF-β1 and IL-2 microspheres were fabricatedusing a double emulsion-evaporation technique. For the TGF-β1microspheres, Poly (lactic-co-glycolic) acid (PLGA-50:50lactide:glycolide, acid terminated) (MW:7,000-17,000) (viscosity:0.16-0.24 dL/g, 0.1% (w/v) in chloroform) (Sigma Aldrich, MO) andPEG-PLGA (PolySciTech, IN) was used to encapsulate rh-TGF-β1 (PeproTech,NJ). Specifically, 170 mg of PLGA and 30 mg of PEG-PLGA was dissolved in4 ml of DCM (Sigma Aldrich, MO). Then 200 μl of aqueous solutioncontaining 10 μg of rh-TGF-β1 was added to the polymer DCM mixture. Themixture was sonicated using a sonicator (Vibra-Cell, Newton, Conn.) for10 sec. at 25% amplitude. Next, this emulsion was then mixed with 60 mlof 2% polyvinyl-alcohol (PVA, MW ˜25,000, 98% hydrolyzed; PolySciences)and homogenized (L4RT-A, Silverson, procured through Fisher Scientific)at 3,000 rpm for 1 min. The homogenized mixtures were then added to 80ml of 1% PVA on stir plate and left for 3 hours in order for the DCM toevaporate. After 3 hours, the microparticles were centrifuged (200 g, 5min, 4° C.), washed 5 times with deionized water, and lyophilized for 48hours (Virtis Benchtop K freeze dryer, Gardiner, N.Y.).

For the IL-2 microspheres, 200 mg of PLGA (PLGA-50:50 lactide:glycolide,acid terminated) (MW:7,000-17,000) (viscosity: 0.16-0.24 dL/g, 0.1%(w/v) in chloroform) (Sigma, Aldrich, Mich.) was combined with 4 ml ofDCM. Subsequently, 5 μg of IL-2 and 150 μl (R&D Systems, MinneapolisMinn.) of deionized water was added to the polymer dissolved in DCM.Next, the mixture was emulsified using a sonicator probe (Vibra-Cell,Newton, Conn.) at 25% amplitude for a period of 25 seconds. Then thisemulsion was mixed with 60 ml of 2% polyvinyl-alcohol (PVA, MW ˜25,000,98% hydrolyzed; Polysciences) and homogenized (L4RT-A, Silverson,procured through Fisher Scientific) at 3,000 rpm for 1 min. Thissecondary emulsion was then then added to 80 ml of 1% PVA on stir plateand stirred for 3 hours. After finishing stirring, the microparticleswere centrifuged (200 g, 5 min, 4° C.), washed 5 times with deionizedwater, and lyophilized for 48 hours (Virtis Benchtop K freeze dryer,Gardiner, N.Y.).

Lastly, the rapamycin (rapa) microspheres were fabricated using a singleemulsion-evaporation technique due to the hydrophobic nature. Rapamycin(Sigma Aldrich, MO) was dissolved in DMSO (Sigma, Aldrich, MO) at 10mg/ml. Then 200 mg of PLGA (Sigma Aldrich, Mich.) was dissolved in 4 mlof DCM. Next, 100 μl of rapamycin (10 mg/ml) was added to thepolymer/DCM mixture. The solution was then homogenized with 60 ml of 2%PVA at 3,000 rpm for 1 min. After homogenizing, the emulsion was thenadded to 80 ml of 1% PVA and stirred for 3 hours. At the end ofstirring, the microspheres were washed 5 times with deionized water andlyophilized for 48 hours.

Characterization of Microspheres—The morphology of the microspheres werecharacterized using scanning electron microscopy (JEOL, JSM-6330F,Peabody, Mass.) and volume impedance measurements were performed on aBeckman Coulter Counter (Multisizer-3, Beckman Coulter, Fullerton,Calif.). The release assay of the IL-2, TGF-β1, and rapamycin wascompleted by incubating 10 mg of microspheres in 1 ml of phosphatebuffered saline (PBS) and 1% BSA, which was placed onto a rotator at 37°C. The supernant was sampled at different time intervals and the TGF-β1and IL-2 release profiles were quantified using an enzyme-linkedimmunosorbent assay (ELISA) (R&D Systems, Minneapolis, Minn.). Therelease profile of the rapamycin microspheres were characterized usingUV-vis spectroscopy and the release media contained 0.2% Tween-80 in PBS(absorbance at 278 nm).

Mice—Female Balb/c mice aged 6-8 weeks were used in this experimentalstudy. (Charles Rivers Laboratories, Wilmington, Mass.). TheInstitutional Animal Care and Use Committee, University of Pittsburghapproved all murine experiments.

Murine DED model and treatment—Dry eye disease was induced using 10mg/ml of Concanavalin A (ConA) (Sigma Aldrich, St. Louis, Mo.) inphosphate buffered saline solution (PBS) was injected (30 μl) into thelacrimal glands with a 28.5 gauge needle using a dissecting microscope.The controls for examining the effects of the TRI MS included Blank(unloaded) or TRI MS (25 mg/ml), which were combined with a PBS solutionof ConA (10 mg/ml). (Olympus SZX10, Waltham, Mass.).

Suppression of Tregs Via the Administration of Anti-GITR

In order to identify the role of Tregs with the administration of ourpreventative treatment, the function of Tregs were inhibited usinganti-GITR (DTA-1) (BioXCell, Lebanon, N.H.) via an intraparietalinjection of (500 μg per mouse) 1 day after injecting the ConA and TRIMS.

Tear Production—Phenol red cotton threads were utilized to measure tearproduction. (Oasis Medical, San Dimas, Calif.). The thread was placed inthe lateral canthus of the eye for a period of 60 seconds, and theamount of wetting on the thread was measured using a dissectingmicroscope (Olympus SZX10, Waltham, Mass.).

Corneal Fluorescein Staining—Fluorescein stain (1% solution) was appliedto the conjunctival sac. The surface of the cornea was examined using adissecting microscope (Olympus SZX10, Waltham, Mass.). The scoring ofstaining was completed by a masked ophthalmologist, and scored 0 for nostaining, score 1 for a quarter of staining, score of 2 for less than ahalf, score of 3 for half, and 4 for more than half of the eye.

Ocular Histology—At the conclusion of the study, the eyes wereexenterated and fixed in 10% neutral buffered formalin Sections wereprepared at approximately 5 μm and stained with Periodic Acid Schiff(PAS) in order to examine goblet cell density. Histological sectionswere scanned and quantified using a Zeiss Axio Scan. Z1 (Thornwood,N.Y.) and Panoramic Viewer software (3D HISTECH Ltd.).

qRT-PCR—Total RNA was extracted from excised lacrimal glands usingTRI-reagent (Molecular Research Center, Cincinnati, Ohio), andquantified using a NanoDrop 2000 (Thermo Scientific). For the reversetranscriptase assay, 2 μg RNA was converted to cDNA using a QuantiTectReverse Transcription Kit (Qiagen, Valencia, Calif.). Quantitativereal-time PCR was then performed using VeriQuest Probe qPCR Mastermix(Affymetrix, Santa Clara, Calif.), (Thermo Scientific) specific for(IFN-γ:Mm01168134_ml, FAM-MGB dye), (IL-1β:Mm00434228_ml, FAM-MGB dye),and (IL-6:Mm00446190_ml, FAM-MGB dye) (Gusb: Mm01197698_ml, VIC-MGB PLdye, endogenous control). Duplex reactions (target gene+GUSB) were runand analyzed on a StepOnePlus Real-Time PCR System (Applied Biosystems,Carlsbad, Calif.). Relative fold changes of IFN-γ, IL-6 and FoxP3expression were calculated and normalized based upon the 2^(ΔΔCt)method, with the saline group as the untreated control.

Immunofluorescences of the lacrimal gland—At the end of the study,lacrimal glands were excised from the mice. Lacrimal glands were fixedwith 4% PFA overnight, followed by cryoprotection through incubation in30% sucrose overnight, and lastly embedded in O.C.T. medium. Thecryosections were obtained at 7 μm thick and stained with fluorescentantibodies. Specifically, 7 μm sections were blocked with 5% normaldonkey serum and 1% Tween20 in PBS. Blocked sections were incubatedovernight at 4° C. with rat anti-FoxP3 (FJK-16s; eBio) and rabbitanti-CD3 (SP7, monoclonal rabbit IgG; Abcam, Cambridge, Mass.). Thesections were then incubated with a secondary antibody, Alexa Fluor 594donkey anti-rat IgG (ThermoFisher Scientific Waltham, Mass.) and AlexaFluor 647 donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories,West Grove, Pa.) for 1 hour at room temperature and then mounted usingFluoroshield mounting medium with DAPI (Abcam, Cambridge, Mass.). Theimages were captured using a Zeiss Axio Scanner Z.1.

Statistical Analysis—Data expressed as mean±S.D. Comparisons betweenmultiple treatment groups were performed using one-way ANOVA, followedby Bonferroni multiple comparisons, and p≤0.05 was consideredstatistically significant. The PCR data expressed as mean±SEM wasanalyzed utilizing a t-test with Welch correction, and p≤0.05 wasconsidered statistically significant. Statistical tests were performedusing GraphPad Prism Software 6.0 (GraphPad Prism, San Diego, Calif.)

Results Characterization of TRI Microspheres: IL-2, TGF-β1 and Rapamycin

The TGF-β1 microspheres were formulated to avoid a 20-day initial lagphase of release. The formulation of TGF-β1 microspheres contains aPEG-PLGA diblock copolymer (4 wt %, Mn ˜5 kDa), which acceleratedrelease by increasing matrix swelling, and the ester-terminated PLGAhelped to minimize the electrostatic interactions between the PLGApolymer and the positively charged protein.

After adapting the release of TGF-β1, the surface morphology of themicrospheres were characterized using scanning electron microscopy(SEM). The SEM images show spherical PLGA based rapamycin microsphereswhile on the other hand, the IL-2 microspheres contained pores with ahigh initial burst followed by a slow continuous release for the lengthof the experimental study. Additionally, the newly fabricated TGF-β1microspheres contained uneven surface morphology. Lastly, the averagesize of the fabricated TRI MS were examined using a Coulter Counter. Theaverage sizes of the microspheres were 12 μm (rapamycin), 19 μm (IL-2),and 17 μm (TGF-β). (See FIGS. 14A-14I). Once the fabricated TRI MS werecharacterized the microspheres were utilized in an experimentalinflammatory model of DED to determine their efficacy in preventing keyfeatures of the disease.

TRI Microspheres Prevent Key Signs of Dry Eye Disease

To investigate whether TRI MS were capable of preventing key signs ofdry eye disease, aqueous tear secretion, goblet cell density, andcorneal fluorescein staining were examined Concanavalin A (ConA) wasinjected into the lacrimal gland to induce DED, and for TRI MS or BlankMS treatment groups, MS were incorporated in ConA injections (FIG. 9).One week following the administration of ConA and the Blank or TRI MS,phenol red thread testing was performed to evaluate aqueous tearsecretion. The administration of ConA (diseased) significantly reducedtear secretion as compared to an injection of saline (non-diseased)(FIG. 10A). Notably, tear secretion was restored to non-diseased levelsin DED mice treated with TRI MS, while administration of Blank MS(unloaded) had no noticeable effect on tear production in diseased mice(FIG. 10A). To identify whether a single factor or a combination offactors was required to prevent loss of aqueous tear production,responses to treatment with single (ex: IL-2 MS) and double (ex: IL-2 MSand TGF-β1 MS) formulations were examined No individual MS formulation,or combination of two MS formulations, were able to restore tearproduction inhibited by ConA, suggesting that therapeutic efficacyrequired delivery of all three factors.

Due to the prevention of reduced tear secretion utilizing thecombination of all three factors (TRI MS), we sought to investigate akey aspect associated with a healthy tear film. Specifically, anintegral component of the tears known as mucin, which is produced bygoblet cells found in the ocular tissue of the conjunctiva. Examinationof the histological sections of the ocular tissue suggested that therewas a significant loss in the density of Periodic Acid Schiff (PAS)stained goblet cells (pink/purple cells in conjunctiva epithelium layer)in the ConA as compared to the saline group (FIGS. 10B, 10C). Moreover,treatment with TRI MS led to maintenance of goblet cell density, unlikemice with ConA-induced DED (with or without Blank MS) (FIG. 10B).Overall, histological sections revealed that TRI MS treatment markedlyinhibited the attenuation of goblet cells that generally contributes toan unstable tear film. Since aqueous tear and mucin production protectsthe corneal epithelium, it was determined whether the restoration oftear production and protection of mucin-producing goblet cells with TRIMS treatment also protected the integrity of the ocular surface.

A healthy tear film has shown to correlate to the maintenance of theocular surface. For this reason, ocular surface health was assessedusing fluorescein staining, with the degree of punctate staining as anindicator of disease severity. Fluorescein images of the ocular surfacewere captured and scored by a masked ophthalmologist on a scale of 0 to4, with 0 corresponding to no staining, and 4 corresponding to stainingon more than 50% of the cornea, as seen in FIG. 11A. The ocular stainingscore was significantly lower for the saline and TRI MS groups ascompared to the ConA and Blank MS groups (FIG. 11B). Eyes from micetreated with ConA plus single or double MS formulations were alsoexamined. The representative scores suggest that there was a significantreduction of fluorescein staining for the rapamycin and TGF-β1microspheres as compared to merely administering IL-2 microspheres.However, the single and double controls were unable to reduce ocularstaining to the same extent as the TRI MS treatment. Collectively, thedata suggest that the local administration of TRI MS prevented a loss ofaqueous tear secretion, maintained goblet cells, and decreased ocularstaining, consistent with amelioration of DED.

TRI MS Decrease Pro-Inflammatory Cytokines and Infiltration of TLymphocytes

The local milieu of the lacrimal glands was examined to identify whetherthe TRI MS altered the underlying immune response that ultimatelyprevented symptoms of DED. Specifically, the levels of pro-inflammatorycytokines was investigated. Notably, there was a significant reductionof IFN-γ, IL-6, and IL-2 in the lacrimal gland of the TRI MS group,compared to the ConA (FIG. 12). The increase of pro-inflammatorycytokine expression correlated to an increase of CD4⁺ T cells in thediseased groups as compared to the TRI MS treated group (FIG. 13).Together this data suggests that the TRI MS treatment was able toinhibit the ConA-induced pro-inflammatory microenvironment in thelacrimal gland tissue.

Suppression of Tregs Via Administration of Anti-GITR

To confirm that Tregs were involved in suppressing the signs associatedwith DED, an agnostic antibody (DTA-1) specific for GITR (glucocorticoidtumor necrosis factor) was administered 1 day after the injection of theConA and TRI MS. Monoclonal antibody anti-GITR (DTA-1) acts tosystemically attenuate the suppressive function of Tregs by inhibitingthe ability of conventional T cells to be suppressed by Tregs. Miceadministered anti-GITR, prior to ConA and TRI MS, developed keypathological features of dry eye disease as indicated by the decrease ofaqueous tear secretion, reduction of goblet cells in the conjunctiva,and the increase in fluorescein staining as compared to the TRI MSgroup. Moreover, with the administration of anti-GITR, levels ofpro-inflammatory cytokines were significantly increased as compared tothe TRI MS. Overall, anti-GITR was utilized to suppress the function ofTregs, and negated any therapeutic effects seen with TRI MS alone. Thisevidence could potentially suggest that the therapeutic effects of TRIMS would be dependent on functional Tregs.

TRI MS Reduce the Proliferation of T-Cells within the Lacrimal Gland

In order to examine the local immune environment of T-cells in thelacrimal gland, immunofluorescence staining on lacrimal glands wascompleted in order to identify CD3⁺ T cells in the tissueImmunofluorescence staining with anti-CD3 and Foxp3 monoclonalantibodies was performed on cryosections of lacrimal glands on thediseases, non-diseased, blank microspheres and TRI MS groups. CD3⁺ Tcells were observed in the lacrimal gland of the diseased, blankmicrospheres and TRI MS groups. Representative images suggest that thereis an overall lower amount of T-cells in TRI MS as compared to diseasedgroup, suggesting that the TRI MS are restoring the immunologicalhomeostasis in the lacrimal gland.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention.

What is claimed is:
 1. A method for treating an ocular disorder in asubject comprising administering a therapeutic agent-loaded carrier toan ocular site of the subject in need thereof, wherein the therapeuticagent loaded-carrier provides controlled delivery of the therapeuticagent under conditions suitable for recruiting regulatory T cells to anocular region of interest or inducing regulatory T cells in an ocularregion of interest.
 2. The method of claim 1, wherein the regulatory Tcells are endogenous.
 3. The method of claim 1, wherein the oculardisorder is dry eye disease, uveitis, allergic conjunctivitis,scleritis, or Age-Related Macular Degeneration (AMD).
 4. The method ofclaim 1, wherein the therapeutic agent is selected from at least one ofCCL22, interleukin 2, rapamycin, transforming growth factor beta(TGF-β), retinoic acid, or vasoactive intestinal peptide (VIP).
 5. Themethod of claim 1, wherein the method comprises recruiting regulatory Tcells to an ocular region of interest, the ocular disorder is dry eyedisease and the therapeutic agent is CCL22.
 6. The method of claim 1,wherein the therapeutic agent-loaded carrier is in the form oftherapeutic agent-loaded microparticles.
 7. The method of claim 1,wherein the therapeutic agent-loaded carrier comprises therapeuticagent-loaded microparticles.
 8. The method of claim 6, wherein themicroparticles comprise poly (lactic-co-glycolic acid).
 9. The method ofclaim 1, wherein the method provides controlled release under conditionsto provide a concentration gradient within or near the ocular disordersite.
 10. The method of claim 6, wherein the therapeutic agent-loadedmicroparticles are included in a composition that does not include ahydrogel.
 11. The method of claim 5, wherein the therapeuticagent-loaded carrier is in the form of therapeutic agent-loadedmicroparticles.
 12. The method of claim 11, wherein the microparticlescomprise poly (lactic-co-glycolic acid).
 13. The method of claim 1,wherein the method comprises recruiting regulatory T cells to an ocularregion of interest.
 14. The method of claim 1, wherein the methodcomprises inducing regulatory T cells in an ocular region of interest.15. The method of claim 1, wherein the therapeutic agent is transforminggrowth factor beta (TGF-β), the therapeutic agent-loaded carrier is inthe form of therapeutic agent-loaded microparticles, and themicroparticles comprise a polyethylene glycol-poly(lactic-co-glycolicacid) copolymer.
 16. The method of claim 1, wherein the therapeuticagent-loaded carrier is not administered with a hydrogel.
 17. The methodof claim 1, wherein the therapeutic agent is selected from CCL22,interleukin 2, rapamycin, transforming growth factor beta (TGF-β),retinoic acid, or vasoactive intestinal peptide (VIP).
 18. The method ofclaim 1, comprising co-administering transforming growth factor beta(TGF-β), interleukin 2, and rapamycin to the subject.
 19. The method ofclaim 18, wherein the transforming growth factor beta (TGF-β)-loadedcarrier is in the form of transforming growth factor beta (TGF-β)-loadedmicroparticles that comprise a polyethyleneglycol-poly(lactic-co-glycolic acid) diblock copolymer and anester-terminated poly(lactic-co-glycolic acid); the interleukin 2-loadedcarrier is in the form of interleukin 2-loaded microparticles thatcomprise poly(lactic-co-glycolic acid); and the rapamycin-loaded carrieris in the form of rapamycin-loaded microparticles that comprisepoly(lactic-co-glycolic acid).
 20. The method of claim 1, wherein thetherapeutic agent is selected from at least one of rapamycin,transforming growth factor beta (TGF-β), or retinoic acid.
 21. A methodfor treating an ocular disorder in a subject comprising administering atherapeutic agent-loaded carrier to an ocular site of the subject inneed thereof, wherein the therapeutic agent loaded-carrier providescontrolled delivery of the therapeutic agent, the therapeutic agent isselected from CCL22, interleukin 2, rapamycin, transforming growthfactor beta (TGF-β), retinoic acid, or vasoactive intestinal peptide(VIP), and the ocular disorder is dry eye disease, uveitis, allergicconjunctivitis, scleritis, or Age-Related Macular Degeneration (AMD).22. A method for treating an ocular disorder in a subject comprisingadministering a therapeutic agent-loaded carrier to an ocular site ofthe subject in need thereof, wherein the therapeutic agentloaded-carrier provides controlled delivery of the therapeutic agentunder conditions suitable for activating regulatory T cells in an ocularregion of interest.
 23. A method for treating an ocular disorder in asubject comprising administering a therapeutic agent-loaded controlledrelease composition to an ocular site of the subject in need thereof,wherein the composition comprises: (a) transforming growth factor beta(TGF-β); (b) interleukin 2; and (c) rapamycin, wherein the compositioncomprises therapeutic agent-loaded microparticles, and the transforminggrowth factor beta (TGF-β)-loaded microparticles comprise a polyethyleneglycol-poly(lactic-co-glycolic acid) diblock copolymer and anester-terminated poly(lactic-co-glycolic acid).
 24. The method of claim23, wherein the ocular disorder is dry eye disease, uveitis, allergicconjunctivitis, scleritis, or Age-Related Macular Degeneration (AMD).